The term scanning microscope as employed in the present invention refers to microscopes in which the object to be imaged is scanned with an illumination pattern. This scanning procedure is commonly carried out in a meandering pattern, so that the object is scanned with the illumination pattern in a manner that is similar, for example, to the way an electron beam is directed onto the screen of a cathode ray tube.
Particularly in biomedical applications, very special scanning microscopes, namely, confocal scanning microscopes, have been in use for quite some time now whenever there is a need for a better resolution along the optical axis than can be obtained with conventional incident-light or transmitted-light microscopes. Regarding the configuration and possibilities of use of confocal scanning microscopes, mention is hereby made, for example, of the literature reference titled “Handbook of biological confocal microscopy”, editor: J. Pawley, Plenum Press, 1995.
Within the scope of German patent applications DE 43 30 347 A1 and DE 199 02 625 A1, a possibility was found of replacing the detection device of a conventional confocal scanning microscope with a spectrally selective detection device that can be adjusted in an almost infinitely variable manner. For this purpose, the inflexible color or interference filters positioned in front of the detectors of conventional confocal scanning microscopes are replaced through the use of an appropriate device for selecting and detecting at least two spectral regions of a light beam. Here, the light beam is first spectrally split by a prism, an optical diffraction grating or a hologram. Then, mirror diaphragms arranged so as to be movable are used to select a first spectral region from the light that has been spectrally split and this first spectral region is then detected by means of a first detector. The light that has not been selected and that strikes the mirror diaphragms is reflected for purposes of detection with a second detector. Hence, the devices described in German patent applications DE 43 30 347 A1 and DE 199 02 625 A1 disclose the possibility of dispensing with the filters that are positioned in front of the detectors and that are inflexible in terms of the possibility of spectral adjustment.
DE 199 06 757 A1 discloses an optical arrangement with which the dichroitic or multichroitic beam splitters of a confocal scanning microscope can be replaced. Here, for purposes of illuminating the object, a spectrally selective element that can be adjusted in an almost infinitely variable manner selects light that comes from the laser light source and that has at least one wavelength and the light that comes from the laser light source and that is reflected and/or scattered on the object is masked out of the detection beam path. An active optical component that can be actuated and that is configured, for example, in the form of an acousto-optical-tunable filter (AOTF) or an acousto-optical deflector (AOD) is employed as the spectrally selective element.
Particularly in the case of the confocal scanning microscopy of weakly fluorescent specimens, the light beam that serves to illuminate the object has an overly short dwell period per scanned object point. Consequently, as a rule, the signal-to-noise ratio of the detected object point is too small or, in fact, even unusable. Especially for physiological applications, there is a great need to examine living specimens. By nature, fast movements occur here and these can only be meaningfully detected if the detected images of the object can be recorded rapidly enough. Such a high detection speed normally likewise yields an insufficient signal-to-noise ratio of the detected image data.